Synthesis of neutral ether lipid monoalkyl-diacylglycerol by lipid acyltransferases

Article abstract of DOI:10.1194/jlr.M073445

In mammals, ether lipids exert a wide spectrum of signaling and structural functions, such as stimulation of immune responses, anti-tumor activities, and enhancement of sperm functions. Abnormal accumulation of monoalkyl-diacylglycerol (MADAG) was found in Wolman's disease, a human genetic disorder defined by a deficiency in lysosomal acid lipase. In the current study, we found that among the nine recombinant human lipid acyltrans-ferases examined, acyl-CoA:diacylglycerol acyltransferase (DGAT)1, DGAT2, acyl-CoA:monoacylglycerol acyltransferase (MGAT)2, MGAT3, acyl-CoA:wax-alcohol acyltransferase 2/MFAT, and DGAT candidate 3 were able to use 1-monoalkylglycerol (1-MAkG) as an acyl acceptor for the synthesis of monoalkyl-monoacylglycerol (MAMAG). These enzymes demonstrated different enzymatic turnover rates and relative efficiencies for the first and second acylation steps leading to the synthesis of MAMAG and MADAG, respectively. They also exhibited different degrees of substrate preference when presented with 1-monooleoylglycerol versus 1-MAkG. In CHO-K1 cells, treatment with DGAT1 selective inhibitor, XP-620, completely blocked the synthesis of MADAG, indicating that DGAT1 is the predominant enzyme responsible for the intracellular synthesis of MADAG in this model system. The levels of MADAG in the adrenal gland of DGAT1 KO mice were reduced as compared with those of the WT mice, suggesting that DGAT1 is a major enzyme for the synthesis of MADAG in this tissu. Our findings indicate that several of these lipid acyltransferases may be able to synthesize neutral ether lipids in mammals.

Full text of DOI:10.1194/jlr.M073445

Synthesis of Neutral Ether Lipid Monoalkyl-diacylglycerol (MADAG)
by Lipid Acyltransferases
Zhengping Maa, Joelle M. Onoratob, Luping Chena,
David W. Nelsonc, Chi-Liang Eric Yenc, Dong Chenga*
From the Departments of Fibrosis Discoverya,
Bioanalytical and Discovery Analytical Sciencesb
Research & Development, Bristol-Myers Squibb Company,
Princeton, NJ 08543-5400a,b
and
Department of Nutritional Sciences, University of Wisconsin-Madison
Madison, WI, 53706c
Running Title: Acylation of monoalkylglycerol by lipid acyltransferases
*Send Proofs and Correspondences to:
Dr. Dong Cheng
Research and Development
Bristol-Myers Squibb Company
PO Box 5400
Princeton, NJ 08543-5400
Tel:
(609)-818-5480
E-mail: dong.cheng@bms.com
1

1Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; AGPS, alkylglycerone
phosphate synthase; AWAT, acyl-CoA:wax-alcohol acyltransferase; CE, cholesteryl ester;
DAG, diacylglycerol; DC, DGAT candidate; DGAT, acyl-CoA:diacylglycerol
acyltransferase; FA, fatty acid; HPLC, high performance liquid chromatography;
MADAG, monoalkyl-diacylglycerol; 1-MAkG, 1-monoalkylglycerol; MAMAG,
monoalkyl-monoacylglycerol; MGAT, acyl-CoA:monoacylglycerol acyltransferase; 1-
MOG, 1-monooleoylglycerol; MS, mass spectrometry; TAG, triacylglycerol; TLC, thin
layer chromatography.
2

Abstract
In mammals, ether lipids exert a wide spectrum of signaling and structural
functions such as stimulation of immune responses, anti-tumor activities and enhancement
of sperm functions. Abnormal accumulation of monoalkyl-diacylglycerol (MADAG) was
found in Wolman’s disease, a human genetic disorder defined by a deficiency in lysosomal
acid lipase. In the current study, we found that among the 9 recombinant human lipid
acyltransferases examined, DGAT1, DGAT2, MGAT2, MGAT3, AWAT2/MFAT and
DC3 were able to use 1-monoalkylglycerol (1-MAkG) as an acyl acceptor for the synthesis
of monoalkyl-monoacylglycerol (MAMAG). These enzymes demonstrated different
enzymatic turnover rates and relative efficiencies for the first and second acylation steps
leading to the synthesis of MAMAG and MADAG, respectively. They also exhibited
different degrees of substrate preference when presented with 1-monooleoylglycerol
versus 1-MAkG. In CHO-K1 cells, treatment with DGAT1 selective inhibitor XP-620
completely blocked the synthesis of MADAG, indicating that DGAT1 is the predominant
enzyme responsible for the intracellular synthesis of MADAG in this model system. The
levels of MADAG in the adrenal gland of DGAT1 knockout (KO) mice were reduced as
compared to those of the wild type mice, suggesting that DGAT1 is a major enzyme for
the synthesis of MADAG in this tissue. Our findings indicate that several of these lipid
acyltransferases may be able to synthesize neutral ether lipids in mammals.
3

INTRODUCTION
Ether lipids constitute a lipid class structurally similar to glycerolipids but
containing ether linkages instead of ester bonds on the glycerol backbone. Ether lipids are
widely distributed in nature and have been found in bacteria (1), deep-sea holothurians (2),
fish (3) and mammals, including humans (4).
In mammals, alkylglycerols are found in high levels in bone marrow and spleen
tissue (4). Injection of exogenous alkylglycerols in vivo and treatment of macrophages in
vitro were shown to stimulate immune responses (5). Alkylglycerols were also suggested
to be useful as adjuvants for boosting the production of vaccines (6).
Natural and synthetic alkylglycerols have been shown to have anti-tumor and anti-
metastatic activities (7, 8). Paradoxically, ether lipids are synthesized at high levels across
many aggressive human cancer cells and primary tumor types through the up-regulation of
alkylglycerone phosphate synthase (AGPS), a critical enzyme for the synthesis of ether
lipids (9). These seemingly confounding effects of exogenously added vs. endogenously
synthesized alkylglycerols suggest the importance of conversions between different ether
lipid species.
In 1976, Lin et al reported the accumulation of neutral ether lipids in the adrenal,
liver and spleen of a male Chinese infant with Wolman’s disease, a rare genetic disorder
caused by a deficiency in lysosomal acid lipase (10). Specifically, increases were found in
monoalkyl-diacylglycerol (MADAG) levels but not in ether lipid species containing
phosphorus. In contrast, in Niemann-Pick disease, another lysosomal storage disease, ether
lipid levels were found to be normal, suggesting that accumulation of neutral ether lipids
might be a characteristic feature of Wolman’s disease (10). More recently, Bartz et al
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quantitatively determined that in lipid droplets isolated from a line of cultured cells,
MADAG accounts for approximately 10–20% of the total neutral lipid pool (11).
Despite emerging evidence that MADAG may play a (as of yet undefined) role in
various disease states, enzymes catalyzing their synthesis are not known. Acyltransferases
of the acyl-CoA:cholesterol acyltransferase (ACAT) family and acyl-CoA:diacylglycerol
acyltransferase 2 (DGAT2) family use lipid molecules such as cholesterol,
monoacylglycerol (MAG), diacylglycerol (DAG), and fatty alcohol as acyl acceptors to
produce, respectively, cholesteryl esters, diacylglycerol (DAG), triacylglycerol (TAG),
and wax esters with varying degrees of specificity and selectivity (12-21). With the
exception of DGAT candidate 3 (DC3), all recombinant enzyme activities have been
reported. Although ACAT enzymes have been shown to acylate oxysterols in addition to
cholesterol, they appear to have no recognition of substrates with long alkyl chains as acyl
acceptors (14, 22). In contrast, DGAT1, and acyl-CoA:wax-alcohol acyltransferase 2
(AWAT2), a member of DGAT2 family, appear to recognize a wide range of substrates,
including MAG, DAG, fatty alcohol and retinol (21, 23, 24). The structural resemblance
between acylglycerols and alkylglycerols suggests a potential shared metabolic route of
synthesis by members of the ACAT and DGAT2 gene family. The current studies are
designed to explore this hypothesis.
Materials and Methods
Materials - Recombinant Flag epitope-tagged human lipid acyltransferases were
expressed in Sf9 or in High5 insect cells using baculovirus system. The recombinant
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enzymes were prepared as previously described (18). 1-Monoalkylglycerol (1-MAkG) was
synthesized in-house (see below). All other chemicals and materials were obtained from
commercial sources: [14C]-oleoyl CoA and [14C]-oleic acid (Perkin-Elmer); 1-
monooleoylglycerol (1-MOG) (Sigma); cell culture tested albumin conjugated oleic acid
(Sigma); electrophoresis reagents (Life Technologies); monoclonal anti-Flag M2 antibody
(Sigma); IRDye 800 goat anti-mouse IgG (Rockland); silica gel thin layer chromatography
(TLC) plates (Whatman 150A).
Synthesis of 1-MAkG - (Z)-1-(octadec-9-enyl)glycerol (1) (Figure 1), a
representative 1-MAkG, was prepared by alkylation of racemic solketal (Sigma) with cis-
9-octadecenyl methanesulfonate (Aldrich) in the presence of sodium hydride in N,N-
dimethylformamide, followed by acid catalyzed hydrolysis (see supplemental Figure 1for
the synthetic details).
LC-MS characterization of MAMAG and MADAG - High resolution mass
spectrometry (MS) was performed on a Thermo-Fisher Q-Exactive mass spectrometer
fitted with a HESI source and coupled to an Accela uHPLC pump and DLW Pal
autosampler. LC was performed on a Waters Symmetry C8 30x2 mm column heated to 65
oC. Mobile phase A consisted of 5 mM ammonium acetate in 95:5 water:methanol, mobile
phase B was 5 mM ammonium acetate in 5:95 water:methanol. Gradient LC was
performed at a flow rate of 0.8 mL/minute starting at 80% B to 100% B in 1 minute then
holding for 10 minutes. MS was performed in positive ionization mode monitoring
primarily the ammonium adducts of the alkylglycerides. MS parameters were as follows:
o
resolution 35,000, source voltage 4200V, capillary temperature 275 C, sheath gas 60,
o
auxiliary gas 30, spare gas 5, probe heater 450 C, S-lens RF 25. MS-MS spectra were
6

acquired using step-collision energy of 10, 20 and 30. Software control was through
Xcalibur.
Reactions with naïve High 5 or MGAT3 microsome membranes using 1-MAkG
and oleoyl CoA as substrates were conducted as described in “Acyltransferase enzyme
reaction” below, except [14C]-oleoyl CoA was excluded from the reaction mix. Liquid-
liquid extracts were reconstituted in 200 µl of methanol:isopropylalcohol:mobile phase A
(60:20:20) and analyzed by LC/MS as described above.
Quantitative immunoblot blot analyses of recombinant acyltransferase expression
levels – Aliquots of 1 µg recombinant acyltransferase microsome membranes used in the
current study were mixed with reducing Laemmli sample buffer and subjected to SDS-
PAGE electrophoresis in a 4-12% Bis-Tris gradient gel. Proteins were transferred to
nitrocellulose membrane using iBlot dry blotting system. The blot was probed with mouse
monoclonal anti-Flag M2 antibody, IRDye 800 goat anti-mouse IgG secondary antibody.
The immunoblot signal was detected and quantified using an Odyssey infrared imaging
system. The anti-Flag signal against Flag-DGAT1 was designated as 1 Protein Unit. The
relative expression levels of all other acyltransferase were quantified using Flag-DGAT1
as the reference.
Acyltransferase enzyme reaction – All acyltransferase enzyme reactions were
carried out in 16x100 screw cap glass tubes (VWR) in 150 mM KHxPO4, pH 7.4 buffer.
For each 200 µl reaction, 180 µl of 0.11 mM 1-MOG or 1-MAkG (final concentration 0.1
mM in the assay) and 10 µl of 3 mg/ml recombinant acyltransferase membranes (30 µg per
assay) were added. Enzyme reactions were initiated by adding 10 µl of 1 mM [14C]-oleoyl
CoA (final concentration 0.05 mM, specific activity 2 µCi/µmol). The reaction tubes were
7

vortexed quickly and incubated in a 37oC water bath for 15 minutes. The reactions were
quenched with 4 ml of stop solution [chloroform : methanol = 2 : 1 (v : v)] and 1 ml water.
The tubes were capped, vortexed and centrifuged at 1000 rpm for 15 minutes at room
temperature to facilitate phase separation (Sorvall RT6000). The aqueous upper phase was
aspirated and the lipid products in the lower phase were dried under nitrogen, reconstituted
with 70 µl hexane and applied for TLC analysis. A radioactive marker lipid standard mix
([14C]-CE, [14C]-TG and [14C]-palmitic acid) was also loaded. TLC was developed in a
pre-equilibrated tank using running solution [hexane : ether : acetic acid = 136 : 24 : 0.8
(v : v : v)] for 40 minutes. Air dried TLC plates were exposed overnight onto a Phosphor
Imager screen and the products were analyzed by the Phosphor Imager (GE Healthcare).
Membranes prepared from Sf9 cells expressing human MGAT1 did not exhibit expected
MGAT activity using 1-MOG as substrate and thus was excluded from further analyses.
Cell-based neutral lipid synthesis in CHO-K1 cells – On Day 0, CHO-K1 cells were
plated in 6-well cell culture plate at a density of approximately 300,000 cells/well. After
overnight incubation at 37oC, 5% CO2, on Day 1, the cells were starved in serum free media
(SFM) for 3 hours. SFM was then replaced by 1.5 ml of reaction mix (0.4 mM oleic acid
- Albumin made in SFM) and incubated at 37oC, 5% CO2 incubator for 30 minutes.
Subsequently 75 µl of label mix (0.5 mM [14C]-oleic acid made in reaction mix with
specific activity at 200 µCi/µmol; final concentration 0.4 mM [14C]-oleic acid in assay with
specific activity at 10 µCi/µmol) was added into each well and incubated for another 1
hour. The labeling materials were then aspirated, cells were washed twice with 2 ml PBS,
and lipid products were extracted twice with 1.5 ml of extraction buffer [hexane :
isopropanol = 3 : 2 (v : v)] for 5 minutes/each. The pooled lipid extracts were dried under
8

nitrogen, reconstituted with 70 µl of hexane and developed with TLC procedure as
described above.
Tissue Lipid Analysis from Wild Type and DGAT1 (-/-) Mice – Mouse husbandry
and tissue collection procedures were approved by the University of Wisconsin–Madison
Animal Care and Use Committee and were conducted in conformity with the Public Health
Service Policy on Humane Care and Use of Laboratory Animals. Mouse jejunum and liver
tissues (16 mg) or whole adrenal gland (~ 2.5 – 5 mg) were homogenized in 200 µl of ice
cold 0.1% ammonium acetate and then 1.5 ml of methanol and 5 ml of MTBE (methyl-
tert-butyl-ether) were added subsequently. The mixture was incubated at room temperature
for 1 hr with vigorous shaking. The lipid/water phases were separated by adding 1.25 ml
of water followed by vortexing, incubation at room temperature for 10 min and
centrifugation for 10 min at 1000 X g. The upper lipid phase was transferred into a fresh
glass tube. The lower phase was re-extracted with MTBE:methanol:water at 10:3:2.5
(v:v:v). The upper lipid phases were pooled and dried under nitrogen gas, reconstituted
with 40 µl hexane and used for TLC. To identify the migration position of MADAG, the
acylation reaction product of 1-MAkG and oleoyl CoA using MGAT3 recombinant
membranes was used as a TLC reference standard. TLC plates were developed in a pre-
equilibrated tank using running solution [hexane : ether : acetic acid = 136 : 24 : 0.8 (v : v :
v)] for 50 minutes. The air dried TLC plates were soaked in the staining solution (10%
CuSO4, 8% phosphoric acid) for 2 minutes and air dried for 5 minutes. The plates were
then baked in an oven at 130 oC for 50 minutes. The image of lipid bands on the TLC plate
was captured using the G Box (Syngene).
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Results
Figure 1 illustrates the schematic diagram of acylation reactions of ether lipid 1-
MAkG for the synthesis of MADAG. (Z)-1-(octadec-9-enyl)glycerol was synthesized as
the representative 1-MAkG for all in vitro enzyme reactions as an 18:1 alkyl chain at the
1-position is the most prominent form isolated from tissue and cellular systems (11). The
first acylation at either 2- or 3-position of the hydroxyl groups of 1-MAkG led to the
products 1-monoalkyl-2-monoacylglycerol or 1-monoalkyl-3-monoacylglycerol,
respectively. Neither LC/MS nor TLC differentiates these two isoforms (Figure 2 and 3).
Consequently, we collectively designated these two products monoalkyl-monoacylglycerol
(MAMAG). In the subsequent second acylation, the remaining hydroxyl group of
MAMAG is further acylated to form the final product monoalkyl-diacylglycerol
(MADAG).
High resolution LC/MS was performed on products extracted from naïve High5 or
MGAT3 enzyme reaction as described in Materials and Methods. As commercial
standards for the alkylglyceride products were unavailable, mass spectra were compared
to available purchased mono-, di- and tri-acylglyceride standards. Similar to purchased
acylglyceride standards, the MAMAG and MADAG products were detected as primarily
their ammonium adduct ions. Measured parent ion masses were within +/- 1.2 ppm of
theoretical. Figure 2 shows the extracted ion chromatograms for a Naïve High5 (panel A)
and MGAT3 (panel B) microsome membranes reaction extract, demonstrating formation
of MADAG only occurred in the presence of MGAT3. Trace levels of MAMAG were
detected in the Naïve High5 extract (Panel A); MAMAG in the presence of MGAT3 was
10

qualitatively increased >60-fold over baseline naïve High5 levels. Further confirmation of
structure was determined using high resolution MS/MS fragmentation. The MS/MS
fragmentation patterns observed for MAMAG and MADAG were similar to the MS/MS
fragmentation patterns of di- and triaclyglyeride standards. The measured fragment masses
were within +/- 2 ppm of theoretical.
To quantitatively compare the ability of lipid acyltransferases to use 1-MAkG as
an acyl acceptor, we conducted acyltransferase reactions and quantified products using
TLC analysis. As shown in Figure 3A, compared to naïve Sf9 and High5 membrane
controls, DGAT1, DGAT2, MGAT2, MGAT3, AWAT2/MFAT and DC3 carried out both
acylation steps resulting in synthesis of the final product MADAG. ACAT2 and
AWAT1/DC7 exhibited no ability to synthesize MADAG but showed ability to synthesize
MAMAG. ACAT1 failed to carry out both the first and second acylation reactions. We
do not know if human MGAT1 uses 1-MAkG as a substrate because the recombinant
membrane prepared from Sf9 cells expressing human MGAT1 did not exhibit expected
MGAT activity using 1-MOG as substrate (data not shown).
Because acyltransferases used here were not purified membrane proteins, we
performed quantitative immunoblot analysis to assess relative protein expression levels for
the estimation of their relative catalytic activities (Figure 3B). Anti-Flag-DGAT1
immunoblot signal was arbitrarily designated as 1 Protein Unit and rest of the Flag-tagged
acyltransferases were compared to Flag-DGAT1 as the reference. After normalizing
protein expression levels, the relative catalytic activity was expressed as nmole
products/min/Protein Unit (Table 1).
11

To compare substrate selectivity, we measure the activities of these enzymes
toward 1-MAkG versus the structurally similar acylglycerol, 1-monooleoylglycerol (1-
MOG). The results shown in Table 1 were generated using the same batch of respective
enzyme source and same lot of [14C]-oleoyl CoA; all reactions were carried out in parallel
for comparison with 1-MOG substrate. AWAT2/MFAT and MGAT2 appeared to be the
least selective enzymes in that the levels of TAG/DAG formation and MAMAG/MADAG
formation were very similar, using the two substrates, respectively. DGAT1 and MGAT3
were moderately selective for 1-MOG (ratios of TAG+DAG formation vs
MAMAG+MADAG formation were 4.2X and 3.0X for DGAT1 and MGAT3,
respectively). DGAT2 appeared to have the highest selectivity for 1-MOG; the level of
TAG+DAG formation was approximately 35X fold higher than the formation of
MAMAG+MADAG (Table 1).
XP-620 is a selective DGAT1 inhibitor that has no inhibitory activities against
DGAT2 gene family members when using monoacylglycerol or diacylglycerol as
substrates (23, 25). When 2-MOG was used as a substrate, the XP-620 IC50 values for
human, rat and mouse DGAT1 were determined to be 0.017 ± 0.004, 0.003 ± 0.001, 0.003
± 0.001 µM respectively (25). Figure 4 illustrates an example of a dose dependent
inhibition of XP-620 against human DGAT1 activity using 1-MAkG as a substrate; the
IC50 value was determined to be 0.026± 0.002 µM (n = 2). As expected, no inhibitory
activity of XP-620 was observed for MGAT2, AWAT2/MFAT or DGAT2 at the
concentrations that showed complete inhibition for DGAT1 (Figure 4).
CHO-K1 cells are known to contain high levels of ether lipids (11). Hence, we
used CHO-K1 whole cell system to investigate intracellular ether lipid synthesis by
12

monitoring the incorporation of [14C]-oleic acid into MADAG. In the absence of XP620,
in 1 hour time period, confluent CHO-K1 cells produced approximately 0.10 nmole
MADAG and 0.54 nmole TAG per well of a 6-well plate. XP-620 dose-dependently
inhibited the synthesis of both MADAG and TAG. At high doses, XP-620 completely
inhibited MADAG production, indicating that DGAT1 is the predominant enzyme
catalyzing the synthesis of MADAG in CHO-K1 cells. In contrast, XP-620 inhibition of
TAG synthesis plateaued at ~80%. The remaining ~20% likely occurs through an alternate
mechanism, such as DGAT2. From the dose response curve, the IC50 values for XP-620
were determined to be 0.012 ± 0.0002 µM (n = 2) for MADAG production and 0.011 ±
0.0002 µM (n = 2) for TAG production, respectively (an example is shown in Figure 5).
To further confirm if the inhibition of MADAG synthesis is DGAT1 dependent,
we tested Compound A and B, two compounds with entirely distinct structural motifs
comparing with XP-620 (Supplemental Figure 2). Compound A has potent inhibitory
potency against human DGAT1 (IC50: 0.020 ± 0.012 µM) but with no inhibitory activity
against human DGAT2. Compound B belongs to a same chemical series but has no
inhibitory activity to both human DGAT1 and DGAT2. When these compounds were
tested in CHO-K1 cells, Compound A demonstrated concentration dependent inhibition
of synthesis of MADAG (IC50: 9.5 µM) whereas Compound B showed no inhibition.
For the TAG synthesis, Compound A exhibited partial inhibition but Compound B had no
effect (Supplemental Figure 2).
To investigate the importance of DGAT1 enzyme for the synthesis of MADAG in
vivo, we analyzed lipid extracts from wild type and DGAT1 knockout mice from tissues of
liver, spleen, jejunum, adrenal gland and plasma. In initial studies, LC/MS, developed to
13

monitor for the presence of MADAG18:1/18:1/18:1 in our in vitro assays, was used to
assess MADAG levels in the tissue extracts. Given the lack of commercially available
standards, this in vitro acylation reaction product of 1-MAkG and oleoyl CoA was used as
a reference standard. Unfortunately, the levels of MADAG18:1/18:1/18:1 in mouse tissues
were below detection by LC/MS. This finding was not surprising given that more than 100
endogenous molecular species of MADAG were reported by Bartz et al (11), with
MADAG18:1/18:1/18:1 being a minor species. As monitoring for more than 100 various
species of MADAG in the absence of standards by mass spectrometry was not practical,
we turned to TLC to qualitatively analyze total MADAG species. In Figure 6, lipid extracts
of adrenal gland and jejunum showed a cluster of MADAG bands that co-migrate with
MADAG18:1/18:1/18:1. The quantification of the bands in the TLC indicates that
MADAG in the adrenal gland of DGAT1 KO mice were ~ 24% less than that in WT mice.
In the jejunum, MADAG levels were similar in DGAT1 KO and WT mice. Stainable
MADAG was not detected in liver or spleen tissue (data not shown). These results suggest
that DGAT1 might be a major enzyme for the synthesis of MADAG in the adrenal gland,
while other acyltransfereses, such as MGAT2 and DGAT2, might compensate and catalyze
MADAG synthesis in DGAT1 KO mice.
Discussion
Ether lipids have been shown to exert a wide range of physiological functions
such as stimulation of immune responses, anti-tumor activity and enhancement of sperm
functions. These physiological functions are associated with the structural and signaling
roles played by ether lipids at the cellular level. MADAG are ether lipids that can account
14

for a significant portion of the total neutral lipid pool. However, metabolic enzymes
responsible for the synthesis of MADAG have not been identified. In the current study,
we found that at least 6 lipid acyltransferases from both the ACAT and DGAT2 gene
families were able to use 1-MAkG as an acyl acceptor for the synthesis of MADAG in
vitro. The existence of this redundant system of 6 enzymes that are able to catalyze similar
acylation reactions suggests that synthesis of the neutral ether lipid MADAG is critical for
mammals. Although mice lacking DGAT1 or MGAT2, two enzymes catalyzing MADAG
synthesis in our study, have not been carefully examined for abnormalities in ether lipid
metabolism, their pleiotropic phenotypes suggest that they may function beyond TAG
synthesis (24, 26-30) In addition, the genetic deletion of each of these genes presumably
can be compensated for by the presence of multiple alternate acyltransferases capable of
carrying out similar reactions.
CHO-K1 cells contain high levels of MADAG in their lipid droplets (11). When
[14C]-oleate was used to trace the synthesis of neutral lipids, the generation of MADAG
was readily detected (Figure 5). The DGAT1 selective inhibitor XP-620 inhibited
intracellular MADAG synthesis in a dose dependent manner; at levels above 0.3 µM XP-
620 completely blocked the synthesis of MADAG. In addition, the IC50 values derived
from the inhibition curve of intracellular synthesis of MADAG was in good agreement
with inhibitory curves of intracellular TAG synthesis and in vitro enzymatic inhibition
curves. These data suggest that DGAT1 is the predominant acyltransferase responsible for
the synthesis of MADAG in CHO-K1 cells.
The physiological functions of neutral ether lipid synthesis are currently unclear.
The process is likely involved in the absorption and transportation of dietary ether lipids
15

as well as the regulation and storage of bioactive ether lipids. Marine species such as certain
shark or rat fish contain rich sources of alkylglycerol in their liver oil. Indeed, multiple
beneficial effects have been reported from traditional medicine supplements of dietary shark
liver oil in countries such as Japan, Norway or Iceland (31). DGAT1, MGAT2, MGAT3 are
expressed in high levels in human small intestines. That there is little reduction of
MADAG synthesis in the jejunum of DGAT1 KO mice compared with wild type mice
(Figure 6) is consistent with the hypothesis that multiple acyltransferases that may play
important roles in the absorption of dietary alkylglycerols from sources such as shark liver
oil. When there are excess amounts of ether lipids present (as when supplied from diet)
MADAG may be synthesized and stored in lipid droplets for future use. When ether lipid
levels are low, lipases (such as lysosomal acid lipase) may hydrolyze the ester bonds of
MADAG and liberate alkylglycerols for signaling purposes or to serve as precursors for
the synthesis of phospho ether lipids.
MADAG accumulation in the affected tissues lacking lysosomal acid lipase suggest
that MADAG metabolism is involved in the pathogenesis of Wolman’s disease. Our
current study identifies several enzymes that can catalyze MADAG synthesis. Their
identification could facilitate further investigation in neutral ether lipid metabolism.
16

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16. Yen C. L., and R. V. Farese Jr. 2003. MGAT2, a monoacylglycerol
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21

Acknowledgements
We thank Chongqing Sun for the synthesis of 1-MAkG and Ching-Hsuen Chu for
providing several recombinant acyltransferase enzymes. D. Nelson and C. Yen were
supported partially by funding from the U.S. National Institutes of Health (R01DK088210
to C. Y).
22

Table 1. Summary of products of various acyltransferases using 1-MAkG or 1-MOG as
substrates
Product of 1-MAkG
Product of 1-MOG
(nmol/min/Protein Unit)
(nmol/min/Protein Unit)
(TAG+DAG)/
(MADAG +
MAMAG)
Enzyme
MADAG/
TAG/
MAMAG
MADAG
DAG
TAG
MAMAG
DAG
3.879 ±
0.100
0.064 ±
0.013
ACAT2
DGAT1
DGAT2
MGAT2
MGAT3
0.02
0.326 ±
0.051
0.248 ±
0.035
0.649 ±
0.018
1.776 ±
0.069
0.76
2.7
3137.4
0.3
4.2
35
0.202 ±
0.043
0.981 ±
0.165
0.013 ±
0.013
41.434 ±
3.604
4.86
4.740 ±
0.271
0.733 ±
0.046
7.778 ±
0.213
2.084 ±
0.010
0.15
1.8
3.0
0.115 ±
0.011
0.925 ±
0.103
0.069 ±
0.009
3.042 ±
0.006
8.04
43.9
AWAT1
/DC7
0.383 ±
0.004
0.026 ±
0.004
0.07
AWAT2
/MFAT
0.893 ±
0.082
0.131 ±
0.013
1.082 ±
0.044
0.124 ±
0.005
0.15
0.1
1.2
0.247 ±
0.024
0.105 ±
0.014
DC3
0.43
Data presented are mean ± SEM from 2 to 4 experiments.
23

Figure 1. Schematic diagram of two step acylations of 1-MAkG catalyzed by
acyltransferases.
24

A
B
Naïve High5 + Substrate
High5_MGAT3 + Substrate
100
3e9
3e9
100
1-MAkG substrate
1-MAkG substrate
80
60
40
20
80
60
40
20
m/z
339.2
69.0705
100
80
60
40
20
0
100
80
100
80
97.1016
MAMAG
MAMAG
1.5e8
1.5e8
m/z
265.2
339.2891
60
60
MS/MS
spectra
40
40
111.1170
135.1169
20
20
149.1323
265.2529
100
80
100
80
4e6
4e6
MADAG
MADAG
50
150
250
350
m/z
450
550
650
60
60
40
40
20
20
m/z 603.5
0
1
2
3
4
5
6
7
8
9
10 11
0
1
2
3
4
5
6
7
8
9
10 11
Time (min)
Time (min)
589.5550
100
80
MS/MS
spectra
m/z 589.5
60
40
20
0
603.5348
700
100
200
300
400
500
600
800
900
m/z
Figure 2. High resolution LC/MS extracted ion chromatograms of MAMAG and
MADAG in High5/MGAT3 cell reactions. Lipid products extracted from Naïve High5
(Panel A) or MGAT3 (Panel B) reactions using 1-MAkG as substrate were analyzed by
high resolution LC/MS as described in Materials and Methods. Shown are the extracted
ion chromatograms for the substrate 1-MAkG (top) and products MAMAG (middle) and
MADAG (bottom). Also shown are the high resolution MS/MS spectra for the products
MAMAG and MADAG.
25

26

Figure 3. TLC analysis of various recombinant acyltransferase acylation products
using 1-MAkG as substrate and their relative protein expressions. Aliquots of 30 µg
microsome membranes with recombinant human acyltransferases expression, naïve Sf9
or High5 microsome membranes were used for acyltransferase reactions using 0.1 mM 1-
MAkG and 0.05 mM [14C]-oleoyl CoA (specific activity at 2 µCi/µmol) in 150 mM
KHxPO4, pH 7.4 buffer at 37oC for 15 minutes as described in Materials and Methods
and the resulting enzyme products were analyzed by TLC. Aliquots of 1µg
acyltransferase microsome membranes were subjected to quantitative immunoblot
analysis as described in Materials and Methods. The relative protein expression level of
each acytransferase was reflected by the Flag band intensity and normalized against that
of DGAT1, which was designated as 1 Protein Unit.(A) TLC profile of lipid products.
CE: cholesteryl ester; MADAG: monoalkyl-diacylglycerol; TAG: triacylglycerol; FA:
fatty acid; MAMAG: monoalkyl-monoacylglycerol; DAG: diacylglycerol. * denotes fatty
acid methyl ester (FAME), a nonspecific product formed during lipid extraction and
analysis. (B) Anti-Flag immunoblot for various acyltransferases.
27

120
100
80
60
40
20
0
DGAT1
MGAT2
AWAT2
DGAT2
DGAT1
IC50 = 0.026 μM
0.001
0.01
0.1
1
XP-620 (µM)
Figure 4. XP-620 concentration dependent effect on 1-MAkG acylation by DGAT1,
MGAT2, AWAT2 and DGAT2. Recombinant DGAT1, MGAT2, AWAT2 or DGAT2
were incubated with varying concentrations of XP-620, substrates 0.05 mM 1-MAkG and
0.05mM of [14C]-oleoyl CoA (specific activity at 2 µCi/µmol) in 150 mM KHxPO4, pH
7.4 at 37oC for 15 minutes. For each enzyme, the percent of products in the presence of
each concentration of XP-620 was calculated compared to that of DMSO (100% control).
The dose response curve was plotted and IC50 values were calculated using “Dose-
response variable slope model” in GraphPad Prism program. XP-620 up to 1 µM showed
no inhibition against MGAT2, AWAT2 and DGAT2. In contrast, it inhibited DGAT1
with IC50 of 0.026 µM.
28

B
120
100
80
60
40
20
0
CHO-K1
C
120
100
80
60
40
20
0
CHO-K1
IC50 = 0.012 μM
IC50 = 0.011 μM
0.0001
0.01
1
100
0.0001
0.01
1
100
XP-620 (µM)
XP-620 (µM)
Figure 5. Concentration dependent inhibition of intracellular synthesis of MADAG
in CHO-K1 cells by XP-620. The lipid synthesis of CHO-K1 cells was analyzed in 6-
well plates when cells were at ~90% confluence. The cells were incubated with 0.5 mM
[14C]-oleic acid for 1 hour in the absence or presence of a series of concentrations of XP-
620. The lipid products were extracted and developed in TLC plates. To calculate IC50
29

values, percent of MADAG or TAG formed in the presence of each concentration of XP-
620 was compared to DMSO (100% control). Dose response curves were plotted and
XP-620 IC50 values were calculated for MADAG or TAG using “Dose-response variable
slope” model in GraphPad Prism program. (A) Phospho Image profile of intracellular
synthesis of MADAG and TAG by CHO-K1 cells. (B) XP-620 concentration dependent
inhibition of intracellular synthesis of MADAG in CHO-K1 cells. (C) XP-620
concentration dependent inhibition of intracellular synthesis of TAG in CHO-K1 cells.
30